Means for regulating the expression of human isoforms of ant

ABSTRACT

ARNi which can selectively inhibit expression of an isoform of ANT, characterized in that said ARNi are an ARN duplex, one of the strands being highly homologous to a fragment of ARNm coding for said isoform of ANT.

The invention relates to means for regulating the expression of humanisoforms of ANT, more particularly to interfering RNA (iRNA) duplexesand uses thereof for said regulation, and to the uses of the cDNAsencoding the isoforms.

The adenine nucleotide translocator (ANT) is the most abundant proteinof the inner membrane of mitochondria. ANT has two distinct functions:it is, firstly, responsible for the transport of adenine nucleotidesacross the inner mitochondrial membrane (import of ADP for oxidativephosphorylation; export of ATP to the cytosol for general metabolism).Secondly, ANT plays an essential role during the mitochondrial phase ofapoptosis. This is because ANT can adopt a nonspecific poreconformation, which results in permeabilization of mitochondrialmembranes and in the triggering of cell death (Kroemer & Reed 2000).

The genes encoding ANTs have been cloned in a large number of species,such as yeast, various plants, cows, rats, mice and humans. All thesespecies have several isoforms, and the structure of the genes is highlyconserved, with an organization consisting of 4 exons separated by 3introns. Human ANT exists in three isoforms (ANT1, ANT2 and ANT3)encoded by three different nuclear genes, which have been cloned andsequenced. ANT1 (chromosome 4) is mainly expressed in the heart and theskeletal muscles. A hereditary disease in humans, associated with amutation in ANT1 (substitution of alanine 114 to proline), is known.This disease is progressive external ophthalmoplegia (a rare conditioncharacterized by substantial deletions of the mitochondrial DNA). ANT2(X chromosome) is very weakly expressed in mature tissues. The highestexpression levels for ANT2 are observed in proliferating cells such asmyoblasts and tumor cells. ANT2 is also specifically found in cellstransformed with the SV40 virus, and also the lines devoid ofmitochondrial DNA (rho°). ANT3 (pseudoautosomal region of the X and Ychromosomes) is expressed ubiquitously in all differentiated tissues.

Apoptosis is a process of cell suicide that takes place in three phases:a pre-mitochondrial phase (heterogeneous), a mitochondrial phase(decision to die), and a degradation phase (“putrefaction” of the cell).ANT, a protein inserted into the inner mitochondrial membrane, has theability to form a pore which radically changes the role of themitochondrion: when ANT is in its OPEN PORE state, the mitochondrionbecomes a cell-destruction organ.

The following points have today been established:

-   -   It is possible to kill cells in vitro by inducing the pore        function of ANT (Belzac, Jacotot et al., Cancer Res. 2001        Feb. 15. 61(4):1260-4).    -   It is possible to protect cardiac cells ex vivo (isolated        reperfused heart) by blocking the pore function of ANT (Di Lisa        et al., J Biol Chem. 2000 Nov 9).    -   It is possible to protect neurons in vivo against death        subsequent to cerebral ischemia, by inhibiting ANT (Cao et al.,        J Cereb Blood Flow Metab. 2001 April 21(4):321-333).

ANT is therefore a major control point for apoptosis and is regulated byendogenous proteins such as the Bax (pro-apoptotic) tumor suppressor andthe Bcl-2 (anti-apoptotic) oncoprotein. ANT is also regulated by viralproteins such as Vpr (pro-apoptotic derived from HIV) and vMIA(anti-apoptotic derived from CMV). It is therefore an ideal target forcombating pathological deregulation of apoptosis.

Recent data have revealed that double-stranded RNA (dsRNA) inducesquenching of the expression of genes whose sequence is very homologousto the sequence of one of the two strands of RNA of the duplex. Thisphenomenon, called RNA interference or iRNA, results in degradation ofthe messenger RNAs (Hammond et al., 2001, Sharp, 2001). Tuschl et al.have demonstrated that the introduction into mammalian cells of a21-nucleotide RNA duplex (small interfering RNA or siRNA) results in thespecific inhibition of gene expression (Elbashir et al., 2001). Aftertransfection, the siRNAs act hand in hand with cellular components (theDICER enzyme and the RISC complex) in order to abolish expression of thetarget gene.

The inventors have noted that it is possible to regulate apoptosis fortherapeutic purposes by acting on the level of expression of the humanisoforms of ANT in a selective manner.

In particular, it has been found that iRNAs designed from defined21-nucleotide regions of the coding sequence of each ANT isoform makesit possible to develop duplex iRNAs capable, after transfection, ofselectively abolishing the expression of each isoform.

The aim of the invention is therefore to provide novel products which,when combined with any method for transferring nucleic acids, can beused in human and animal therapy.

The invention is directed toward iRNAs capable of selectively inhibitingthe expression of an ANT isoform, characterized in that said iRNAs arean RNA duplex, one of the strands being highly homologous to a fragmentof the mRNA encoding said ANT isoform.

Advantageously, the iRNAs of the invention are siRNAs (small interferingRNAs) of 18 to 25 nucleotides, more particularly of 21 nucleotides.

Preferred iRNAs are chosen from the duplexes with strands of sequencesSEQ ID No. 1 and SEQ ID No. 2; SEQ ID No. 3 and SEQ ID No. 4; SEQ ID No.5 and SEQ ID No. 6: SEQ ID No. 1: 5′-acagaucagugcugagaagdTdT-3′ SEQ IDNo. 2: 5′-cuucucagcacugaucugudTdT-3′ SEQ ID No. 3:5′-gcagaucacugcagauaagdTdT-3′ SEQ ID No. 4:5′-cuuaucugcagugaucugcdTdT-3′ SEQ ID No. 5:5′-gggcaucguggacugcauudTdT-3′ SEQ ID No. 6:5′-aaugcaguccacgaugcccdTdT-3′

The invention is also directed toward constructs containing at least oneiRNA as defined above or DNA sequences encoding each of the strands ofthese iRNAs.

In one embodiment of the invention, the construct is characterized inthat the iRNA is associated with a vector that facilitates itsadministration, its passage across membranes, tissues or biologicalinteguments, in particular cytoplasmic membranes, mitochondrialmembranes, nuclear membranes, skin, mucous membranes, endothelial walls,the blood-brain barrier, and also its bioavailability, its stability andits pharmacodistribution, such as a peptide, a liposome, nanoparticles(nanospheres, nanotubes), or a non-natural oligomer such as ureapolymers.

In another embodiment, the construct is characterized in that the iRNAis associated with a vector for transferring nucleic acids, such asretroviruses (Barton and Medzhitov, PNAS, 2002, vol. 99 (23): p14943-14945), transposons, adenoviruses (Xia et al.; Nature Bidech,2002, vol. 20, p 1005-1010) or plasmids (Brummelkamp et al., CancelCall, 2002, p 243-247).

The invention is also directed toward the pharmaceutical compositionscharacterized in that they contain an effective amount of at least oneiRNA as defined above, or a construct as defined above, in combinationwith a pharmaceutically acceptable vehicle.

Advantageous pharmaceutical compositions are characterized in that theyare in injectable form.

Other presentation forms are suitable for oral, parenteral, rectal ortopical administration (Levis et al., Nature Genetics, 2002, vol. 32, p107-108).

The iRNAs, constructs or pharmaceutical compositions as defined aboveare characterized in that they have the ability to regulate (to induceor to inhibit) mitochondrial membrane permeabilization and cell death ofapoptotic, necrotic and autophagic type and related mechanisms.

The compositions of the invention make it possible to regulate theexpression of human isoforms of ANT and, in this respect, areparticularly useful for the treatment of pathologies associated withderegulation of apoptosis and other related forms of cell death.

The invention therefore relates, in part, to the use of siRNA-ANT1,siRNA-ANT2 and/or siRNA-ANT3 for inducing/promoting (siRNA-ANT2) or,conversely inhibiting (siRNA-ANT and/or siRNA-ANT3) the drop inmitochondrial transmembrane potential (ΔΨm) and apoptosis and death ofapoptotic, necrotic and autophagic type, and related mechanisms.

The invention therefore also relates to the use of hANT1, hANT2 and/orhANT3 cDNAs for inducing/promoting (hANT1 cDNA and/or hANT3 cDNA) or,conversely, inhibiting (hANT2 cDNA) the drop in mitochondrialtransmembrane potential (ΔΨm) and apoptosis.

Mention is in particular made of their use for treating an apoptosisdeficiency, for example in the various forms of cancer, and autoimmunediseases, such as disseminated lupus erythematosus or arthritis.

In other uses, these compositions are used for treating an excess ofapoptosis, such as, for example, neurodegenerative diseases (Alzheimer'sdisease, Parkinson's disease, Huntington's disease) and cerebral andcardiac ischemias.

For example, ANT1 or ANT3 siRNAs, or alternatively ANT2 cDNA, may beused for inhibiting neuronal death in ischemic situations or situationsof neurodegenerative pathologies, or else for inhibiting cardiomyocytedeath in ischemic situations, or hepatocyte death (viral infections,drug-related poisonings). For example, h-ANT2 siRNAs and/or h-ANT1 orh-ANT3 cDNAs may be used for inducing tumor cell apoptosis orautoreactive lymphocyte apoptosis.

Said pharmaceutical compositions are also of great advantage for thetreatment of HIV infections.

Other characteristics and advantages of the invention will emerge in thesubsequent description, and with reference to FIGS. 1 to 6, whichrepresent, respectively:

FIG. 1. Complete cDNA sequences encoding the three human isoforms ofANT, isolated after RT/PCR using RNAs originating from 293T and HeLacells.

FIG. 2. Expression of the hANT1 isoform and expression of the hANT3isoform induce apoptosis. Flow cytometry analysis of 293T cells, 24hours after cotransfection of 1 μg of vector pIRES-2-eGFP with 1 μg ofvector pcDNA3.1-hANT. The intensity of the CMXRos label is quantifiedonly on the GFP positive cells. B. Flow cytometry analysis of 293Tcells, 24, 48 or 72 hours after transfection with 1 μg of vectorpIRES-2-eGFP or with 1 μg of each vector pIRES-eGFP-hANT. The intensityof the CMXRos label is quantified only on the GFP positive cells. C.Flow cytometry analysis of the frequency of hypoploid nuclei on 293Tcells, 24, 48 or 72 hours after transfection with 1 μg of vectorpIRES-eGFP or 1 μg of each vector pIRES-eGFP-hANT.

FIG. 3. The apoptosis induced by the expression of hANT1 and hANT3 isinhibited by ZVAD and Boc D but not by CsA. A. Flow cytometry analysisof 293T cells 48 hours after transfection with 1 μg of vectorpIRES-2-eGFP or with 1 μg of each vector pIRES-eGFP-hANT in the presenceor absence of 10 μM of CsA. The intensity of the CMXRos label isquantified only on the GFP positive cells. B. Flow cytometry analysis of293T cells 48 hours after transfection with 1 μg of vector pIRES-2-eGFPor with 1 μg of each vector pIRES-eGFP-hANT in the presence or absenceof 100 μM of ZVAD-fmk or of 100 μM of Boc D. The intensity of the CMXRoslabel is quantified only on the GFP positive cells.

FIG. 4. The expression of Bcl2 inhibits apoptosis induced by theexpression of the hANT1 and hANT3 isoforms. HeLa Neo and Bcl2 cells aretransfected with 1 μg of vector pIRES-2-eGFP or with 1 μg of each vectorpIRES-eGFP-hANT and, after 72 hours, the intensity of the CMXRos labelis analyzed by flow cytometry on the GFP positive cells.

FIG. 5. Subcellular localization of the hANT1 and hANT2 isoforms. HeLacells are transfected with 1 μg of vector pcDNA3.1V5-hANT1 (A) or with 1μg of vector pcDNA3.1V5-hANT2 (B) and then fixed with paraformaldehyde.The colocalization of the hANT-V5 fusion proteins with the COXmitochondrial protein is determined by immunofluorescent detection ofthe V5 epitope (green fluorescence) and of the COX protein (redflorescence). The “merge” image represents the superimposition of thegreen fluorescence and red fluorescence showing the colocalization.

FIG. 6. Specific inhibition of the expression of the human isoforms 1and 2 of ANT via the use of specific siRNAs.

-   -   (A) HeLa cells are cotransfected with, firstly, an expression        vector pcDNA3.1V5-hANT1 and, secondly, siRNAs specific for hANT1        or hANT2mut.    -   (B) HeLa cells are cotransfected with, firstly, an expression        vector pcDNA3.1V5-hANT2 and, secondly, siRNAs specific for hANT2        or hANT2mut.

24 hours after transfection, the cells are lyzed and the expression ofthe ANT isoforms is determined by Western blotting using an anti-V5monoclonal antibody.

Cotransfections: HeLa cells are cultured in 6-well plates inDMEM/Glutamax-I supplemented with 10% of fetal calf serum. After 24hours, the cells are transfected by adding 3 μl of lipofectamine 2000(Invitrogen), 3 μg of siRNA and 1 μg of vector pcDNA3.1V5-hANT1 or 2 inserum-free DMEM (final volume of 500 μl). The cells are rinsed 6 hoursafter transfection and maintained in culture for 24, 48 or 72 hours.

Cell extract preparations and Western blotting: The cells areresuspended in 100 μl of lysis buffer (25 mM Tris-HCl, pH 7.5, 25 mMNaCl, 5 mM EDTA, 1% Triton X-100, cocktail of protease inhibitors) andcentrifuged for 10 minutes at 13 000 rpm at 4° C. 10 μl of thesupernatant are collected in order to carry out a Bradford test. Theextracts are then analyzed by SDS-PAGE gel after denaturation for 3minutes at 100° C. in the presence of SDS-Laemmli buffer. Aftertransfer, the proteins are revealed with an anti-V5 antibody (1/5000Invitrogen).

Cloning of the human isoforms of ANT and production of expressionvectors: Total RNA from 293T cells and from HeLa cells was isolated(Trizol protocol) and used in reverse transcription/amplificationexperiments initiated with an oligo dT-type primer. Primers specific forthe human isoforms of ANT (hANT1, hANT2 and hANT3) were synthesizedbased on the sequences published in GenBank in order to specificallyamplify the complete cDNA of each of the isoforms (table 1). Theseproducts were then subcloned into the vector pGEM-T after the additionof a dAdenosine residue at their ends. The sequence of each insert wasverified (FIG. 1). The cDNAs encoding the three isoforms were thencloned into expression vectors: pcDNA3.1 (version +, Invitrogen) andpIRES-2-eGFP (Clontech). To generate fusion proteins with the V5 epitopecorresponding to the three isoforms, an amplification approach (table 2)made it possible to modify the ends of the cDNAs encoding the threeisoforms (mutation of the STOP codon and also addition of restrictionenzyme recognition sequences) and to subclone these products into thevector pcDNA3.1-V5 (version A, Invitrogen). The final constructs wereverified by sequencing.

Apoptotic potential of the human isoforms of ANT: The transfectionexperiments were carried out on 293T cells, using the empty vectorpIRES-2-GFP as a control or the vectors pIRES-2-eGFP containing thesequences of the cDNAs encoding the three isoforms of hANT. At a giventime post-transfection, the cells were analyzed by flow cytometry.

The results show that the expression of the hANT1 and hANT3 isoformsresults in a dissipation of the mitochondrial potential, thus triggeringapoptosis, whereas the expression of the hANT2 isoform does not affectthe mitochondrial integrity (FIG. 2).

Using a similar experimental approach, we demonstrate that the apoptosisassociated with the expression of the hANT1 and hANT3 isoforms isinhibited by caspase inhibitors (ZVAD and Boc D) (FIG. 3A) but not bycyclosporin A (CsA) (FIG. 3B).

We also demonstrate, using HeLa cells overexpressing the Bcl2 protein,that the latter is capable of inhibiting the apoptosis induced by thehANT1 and hANT2 isoforms (FIG. 4).

Subcellular localization of the hANT1 and hANT2 isoforms: Aftertransfection of HeLa cells with constructs encoding the hANT1-V5 andhANT2-V5 fusion proteins, we carried out immunolabeling in order todetermine the subcellular localization of hANT1 and hANT2. The analysisof the localization of the signal obtained with an anti-V5 antibody andthe signal obtained with an antibody directed against COX (amitochondrial protein) demonstrates a mitochondrial localization for thehANT1 and 2 isoforms (FIG. 5).

iRNA Duplex of the Human Isoforms of ANT

Preparation of iRNAs. The double-stranded siRNAs corresponding to thecDNA sequences of human ANT1 (AAACAGATCAGTGCTGAGAAG, nucleotides127-147), human ANT2 (AAGCAGATCACTGCAGATAAG, nucleotides 127-147), humanANT2 containing four mutations (AAGCGGATCGCTACAAATAAG, nucleotides127-147) and human ANT3 (AAGGGCATCGTGGACTGCATT, nucleotides 154-174)were designed according to the recommendations of Elbashir et al.(2001). The duplexes were prepared by Proligo (France). hANT1 (127-147)DNA sequence: 5′-aaacagatcagtgctgagaag-3′ (SEQ ID No. 7) iRNA duplex:5′-acagaucagugcugagaagdTdT-3′ (SEQ ID No. 8)5′-cuucucagcacugaucugudTdT-3′ (SEQ ID No. 9) hANT2 (127-147) DNAsequence: 5′-aagcagatcactgcagataag-3′ (SEQ ID No. 10) iRNA duplex:5′-gcagaucacugcagauaagdTdT-3′ (SEQ ID No. 11)5′-cuuaucugcagugaucugcdTdT-3′ (SEQ ID No. 12) hANT2mut (127-147) DNAsequence: 5′-aagcggatcgctacaaataag-3′ (SEQ ID No. 13) iRNA duplex:5′-gcggaucgcuacaaauaagdTdT-3′ (SEQ ID No. 14)5′-cuuauuuguagcgauccgcdTdT-3′ (SEQ ID No. 15) hANT3 (154-174) DNAsequence: 5′-aagggcatcgtggactgcatt-3′ (SEQ ID No. 16) iRNA duplex:5′-gggcaucguggacugcauudTdT-3′ (SEQ ID NO. 17)5′-aaugcaguccacgaugcccdTdT-3′. (SEQ ID No. 18)

The tables hereinafter give, respectively, the sequences of the primersused:

-   -   Table 1: in RT/PCR experiments in order to clone the cDNA        encoding the three human isoforms of ANT.

Table 2. for the construction of the expression vectors containing thecDNAs encoding the hANT-V5 fusion proteins. TABLE 1 Sense primerAntisense primer hANT1 (SEQ ID No. 22 and 23)5′ATGGGTGATCACGCTTGGAGCTTCCTAAAG3′ 5′TTAGACATATTTTTTGATCTCATCATACAA3′hANT2 (SEQ ID No. 24 and 25) 5′ATGACAGATGCCGCTGTGTCCTTCGCCAAG3′5′TTATGTGTACTTCTTGATTTCATCATACAA3′ hANT3 (SEQ ID No. 26 and 27)5′ATGACGGAACAGGCCATCTCCTTCGCCAAA3′ 5′TTAGATCACCTTCTTGAGCTCGTCGTACAG3′

TABLE 2 Sense primer Antisense primer hANT15′TAAGGTACCATGGGTGATCACGCTTGGA3′ (SEQ ID No. 28 and 29)5′ATCTCGAGGACATATTTTTTGATCTC3′ hANT2 5′TAAGGTACCATGACAGATGCCGCTGTGT3′(SEQ ID No. 30 and 31) 5′ATCTCGAGTGTGTACTTCTTGATTTC3′ hANT35′TAAGGTACCATGACGGAACAGGCCATCT3′ (SEQ ID No. 32 and 33)5′ATCTCGTGGATCACCTTCTTGAGCTC3′

REFERENCES

-   Hammond, S. M., Caudy, A. A. and Hannon, G. J. (2001).    Post-transcriptional gene silencing by double-stranded RNA. Nat Rev    Genet, 2, 110-119.-   Sharp, P. A. (2001). RNA interference-2001. Genes Dev. 15, 485-490.-   Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K.    and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA    interference in cultured mammalian cells, Nature, 411, 494-498.

1. An iRNA capable of selectively inhibiting the expression of an ANTisoform, characterized in that said iRNA is an RNA duplex, one of thestrands being highly homologous to a fragment of the mRNA encoding saidANT isoform.
 2. The iRNA as claimed in claim 1, characterized in that itis an siRNA of 18 to 25 nucleotides, more particularly of 21nucleotides.
 3. The iRNA as claimed in claim 2, characterized in that ithas the sequence SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No.
 3. 4. Aconstruct containing at least one iRNA as claimed in claim 1, or DNAsequences encoding each of the strands of these iRNAs.
 5. The constructas claimed in claim 4, characterized in that the iRNA is associated witha vector that facilitates its administration, its passage acrossmembranes, tissues or biological integuments, in particular cytoplasmicmembranes, mitochondrial membranes, nuclear membranes, skin, mucousmembranes, endothelial walls, the blood-brain barrier, and also itsbioavailability, its stability and its pharmacodistribution, such as apeptide, a liposome, nanoparticles (nanospheres, nanotubes), or anon-natural oligomer such as urea oligomers.
 6. The construct as claimedin claim 4, characterized in that the vectors are vectors fortransferring nucleic acids, such as retroviruses, transposons,adenoviruses or plasmids.
 7. A pharmaceutical composition characterizedin that it contains an effective amount of at least one iRNA as claimedin claim 1, or a construct containing at least one of said iRNA or DNAsequences encoding each of the strands of these iRNAS, in combinationwith a pharmaceutically acceptable vehicle.
 8. The pharmaceuticalcomposition as claimed in claim 7, characterized in that it is ininjectable form, or in a form that can be administered orally,parenterally, rectally or topically.
 9. The iRNA as claimed in claim 1,or a construct containing at least one of said iRNA or DNA sequencesencoding each of the strands of these iRNAS, or a pharmaceuticalcomposition containing an effective amount of at least one of the iRNA,characterized in that it has the ability to regulate (to induce or toinhibit) mitochondrial membrane permeabilization and cell death ofapoptotic, necrotic and autophagic type, and related mechanisms.